The invention relates to generation of dendritic cells from expanded populations of monocytes. These dendritic cells are potent antigen presenting cells which can mediate a variety of T cell responses. The invention relates to the fields of immunology, molecular biology, and medicine.
T cells mediate most forms of cellular immunity, including cell lympholysis, delayed type hypersensitivity (DTH), transplantation rejection, and allograft rejection. An introduction to T cells and cell mediated immunity is found in Paul (1993) Fundamental Immunology, Third Edition Raven Press, New York, N.Y. and the references cited therein.
Typical T cells do not respond to free antigenic peptides. Some T cells interact with a specialized set of cell surface proteins (the class I and class II major histocompatibility complexes, or MHC) which present antigens on the surface of cells (T cells also recognize antigens in the context of other molecules). Cytotoxic and helper T cells are induced to proliferate by specialized antigen presenting cells, such as macrophage and dendritic cells, which present antigenic epitopes, such as peptides or carbohydrates, on their cellular surfaces in conjunction with MHC molecules. T cells are induced by these antigen presenting cells to recognize corresponding antigens expressed, e.g., on MHC antigens on the surface of target cells. T cells destroy these target cells, or induce other cells to destroy these target cells.
Certain T cells can recognize the antigen in the form of a polypeptide fragment bound to the MHC class I molecules on target cells, rather than the intact polypeptide itself. The polypeptide is endogenously synthesized by the cell, and a portion of the polypeptide is degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit xcex22 microglobulin. The peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific T cells. Investigations of the crystal structure of the human MHC class I molecule HLA-A2.1 indicate that a peptide binding groove is created by the folding of the xcex11 and xcex12 domains of the class I heavy chain (Bjorkman et al., (1987) Nature 329:506). Falk et al., (1991) Nature 351:290 have developed an approach to characterize naturally processed peptides bound to class I molecules. Other investigators have successfully achieved direct amino acid sequencing of the more abundant antigenic peptides in various HPLC fractions by conventional automated sequencing of peptides eluted from class I molecules (Jardetzky, el al. (1991) Nature 353:326 and mass spectrometry Hunt, et al., Science 225:1261 (1992). A review of the characterization of naturally processed peptides in MHC Class I is found in Rxc3x6tzschke and Falk (1991) Immunol. Today 12:447.
Target T cells recognizing antigenic peptides can be induced to differentiate and proliferate in response, for example, to antigen presenting cells bearing antigenic peptides in the context of MHC class I and class II complexes. There are differences in the antigenic peptides bound to MHC class I and class II molecules, but the two classes of bound peptides share common epitopes within the same protein which enable a T cell activated by an antigen presenting cell to recognize a corresponding epitope in the context of MHC class I or II, or other cell surface molecules. MHC class I molecules on target cells typically bind 9 amino acid antigenic peptides, while corresponding MHC class II-peptide complexes have greater heterogeneity in the size of the bound antigenic peptide.
Dendritic cells are the most potent antigen presenting cells known, being capable of activating T cells, NK cells and other immune cells by presentation of peptides and carbohydrate antigens on the MHC class I and class II molecules on the surface of the cells. An extensive review of the origin, maturation and antigen presenting function of dendritic cells in reviewed in Banchereau and Schmitt (1995) Dendritic Cells In Fundamental and Clinical Immunology Volume 2, in Advances in Experimental Medicine and Biology (Back et al. eds), volume 378 Plenum Press, NY. A short review is found in Cella et al. (1997) Current Opinion in Immunology 9:10-16, and the references cited therein. See also, Hart (1997) Blood 90:3245 (1997); J. Banchereau and R. M. Steinman (1998) Nature 392: 245; Schuler et al. (1997) Int Arch Allergy Immunol 112:317-322; Rescigno et al. (1997) Journal of Leukocyte Biology 61:415-421, Clark (1997) J. Exp. Med. 185(3) 801-803, Sprent (1995) Current Biology 5(10): 1095-1097; Nair et al. (1995) International Immunology 7(4):679-688; Caux et al. (1995) immunology Today 16(1):2-4; Liu et al. (1996) International Review of Cytology 166:139-179, and O""Doherty et al. (1993) J.Exp. Med. 178:1067-1078, and the references cited in each article.
xe2x80x9cImmaturexe2x80x9d and xe2x80x9cmaturexe2x80x9d phenotypic subsets of dendritic cells have been characterized and methods for the isolation and/or generation of DCs have been described, including various conditions for generating xe2x80x9cimmaturexe2x80x9d and xe2x80x9cmaturexe2x80x9d DC subsets. See, e.g., Steinman (1991) Ann Rev Immunol 9:271; Steinman et al. (1993) Adv Exp Med Biol 329:1; Schuler et al. (1997) Int Arch Allergy Immunol 112:317; Jaffe (1993) Pediatric Pathology 13:821. Peters, et al., (1996) Immunology Today 276:273; Herbst, et al. (1996) Blood 88:2541. Romani et al. (1996) J Immunol Methods 196:137. Cella et al. (1997) Current Opinion Immunology 9: 10. Morse, et al. (1997) Annals of Surgery 226:6; Santiago-Schwartz, et al. (1992) J Leuk Biol 52:274; Zhou and Tedder (1996) Proc Natl Acad Sci USA. 93:2588; C. Caux, et al. (1996) J Exp Med 184;695; C. Caux, et al. (1997) Blood 90:14589; Winzler, et al., (1997) J Exp Med 185:317. Sallusto and Lanzavecchia (1994) J Exp Med 179:1109.
A general introduction to the use of dendritic cells for immunotherapy is provided by Girolomoni et al. (1997) in Immunology Today. In addition to presenting antigens to T-cells and NK cells, dendritic cells stimulate T cell mitogenesis, e.g., by producing the T cell mitogen IL-12. See, e.g., Jonuleit et al. (1997) Journal of Immunol. 2610-2614.
Despite the clear value of dendritic cells for immunotherapy, problems remain in using dendritic cells for therapeutic applications. Primarily, dendritic cells are very rare in peripheral blood, making isolation of sufficient numbers of such cells for therapeutic applications impractical. For example, autologous therapies in which dendritic cells are isolated from a patient and loaded with a particular peptide or carbohydrate antigen for T cell activation are impractical in the absence of large numbers of dendritic cells. Accordingly, there exists a need in the art for a method of making dendritic cells which are capable of T cell activation, particularly in the context of autologous therapeutic approaches. This invention solves these and other problems.
The present invention derives, in part, from the surprising discovery that IL-3 cultured expanded populations of monocytes are suitable for in vitro differentiation into dendritic cells. Thus, the present invention overcomes problems of the prior art by providing an easy method of generating large numbers of dendritic cells, i.e., from cultured monocytes. This, in turn facilitates the use of dendritic cells to generate cell-mediated immune responses.
Accordingly, in one embodiment, the invention provides methods of differentiating monocytes into dendritic cells. In the methods, monocytes are incubated in the presence of IL-3, causing the monocytes to proliferate, yielding an expanded population of monocytes. The expanded population of monocytes is differentiated into dendritic cells, e.g., by culturing the expanded population of cells with GM-CSF and IL-4 (to produce baseline or Type I DCs) and, optionally, TNF-xcex1, CD40 ligand, IL-1xcex1 or IL-1xcex2 (to produce xe2x80x9cactivatedxe2x80x9d or xe2x80x9ctype IIxe2x80x9d DCs). For example, when baseline DCs are incubated with TNF-xcex1, an xe2x80x9cactivatedxe2x80x9d or xe2x80x9ctype IIxe2x80x9d dendritic cell, characterized by the expression of certain markers, results. It is surprisingly discovered that the effects of TNF-xcex1 are reversible and reinducible in human DCs. TNF-xcex1 transiently activates DCs to a heightened proinflammatory state. Similar effects are obvserved upon incubation with IL-1xcex1 or IL-1xcex2 and CD40 ligand; in certain applications, any of these cytokines, or a combination thereof, are preferred for making activated DCs. Slightly different phenotypes are observed when different cytokines are used for activation; for example, CD 40 ligand results in expression of IL-12 in DCs.
Monocytes are obtained from a variety of sources, such as leukapheresis of peripheral blood mononuclear cells from a patient, followed by elutriation of the isolated peripheral blood to provide isolated monocytes.
Typically, a peptide is loaded onto the surface of resulting dendritic cells for presentation to a T cell, resulting in proliferation of the T cell (as measured, e.g., in an MLR assay), in vitro or in vivo. Peptide is loaded by any of a variety of methods, including incubation of the peptide with the dendritic cell, incubation of a protein comprising the peptide with DC, transduction of DC (or the progenitor expanded monocyte population) with a gene encoding the peptide (or a protein comprising the peptide), or the like. Typical antigens for use as peptides are derived from those expressed in a target cell such as a transformed cell, a cancer cell, a bacterial cell, a parasitically infected cell or a virally infected cell, or the like. Examples include, but are not limited to, carbohydrates such as mucin, tumor antigens, peptides derived from a protein selected from the group consisting of HIV Gag, HIV Env, HER-2, MART-1, gp-100, PSA, HBVc, HBVs, HPV E6, HPV E7, tyrosinase, MAGE-1, trp-1, mycobacterial antigens, and CEA, as well as many others. Tumor antigens suitable for presentation include, but are not limited to, c-erb-xcex2-2/HER2/neu, PEM/MUC-1, Int-2, Hst, BRCA-1, BRCA-2, truncated EGFRvIII, MUC-1, CEA, p53, ras, RK, Myc, Myb, OB-1, OB-2, BCR/ABL, GIP, GSP, RET, ROS, FIS, SRC, TRC, WTI, DCC, NF1, FAP, MEN-1, ERB-B1 and idiotypic immunoglobulins (e.g., from a B cell of a non-Hodgkin""s lymphoma patient). Ordinarily, the antigens are expressed on the surface of the target cell in the context of an MHC class I molecule (but peptide is also presented in other contexts), which is recognized by the T cell. The antigen presenting activity of dendritic cells is enhanced by co-culture with certain cytokines such as TNF-xcex1 or IL-1xcex1 or IL-1xcex2. In addition, the cytokines produced by dendritic cells (e.g., IL-12) provide therapeutic benefits, by stimulating T cells and NK cells.
In ex vivo therapeutic applications, T cells are isolated from a patient, activated in vitro, and re-introduced into the patient. Similarly, dendritic cells made from monocytes isolated from a patient and expanded in culture are differentiated in vitro into dendritic cells, which are used to activate T cells in vitro, or by re-introduction into the patient.
It will be appreciated that the availability of high numbers of dendritic cells provide methods of activating a T cell. In basic form, the method includes contacting the T cell with a dendritic cell made according to the methods of the invention, e.g., in culture, or in vivo wherein the dendritic cell is made by the method of claim 1, thereby producing an activated T-cell. Activated T cells are useful for the treatment of a variety of disorders, including cancer, viral, bacterial or parasitic infection. Activated T cells are competent to kill tumorigenic or infected cell, or direct a helper response against such cells.
Cell cultures for making recombinant dendritic cells are provided. These cultures include an expanded population of monocytes, and, typically cytokines such as IL-4 and GM-CSF. The cultures optionally include other cytokines such as IL-3 or TNF-xcex1, or IL-1xcex1 or IL-1xcex2. Depending on the mammal from which the cell culture is selected, the cytokines are appropriately derived from the particular species. For example, where the cells are of human origin, hGM-CSF is typically used. Where the cells are of murine origin, mGM-CSF is used. However, it will be recognized that many such proteins are active outside of the particular mammal from which they are derived.
Diagnostic methods and methods for assessing whether a target antigen is an appropriate target for a T cell are provided. For example, T cell mediated anti-cancer cell (or other target cell) activity of a target antigenic peptide is measured in one embodiment. In this embodiment, a dendritic cell is provided from an expanded population of monocytes. The dendritic cell comprises the antigenic peptide. A T cell is contacted with the dendritic cell (from an autologous source), thereby providing an activated T cell with specificity for the antigenic peptide. The cancer cell is contacted with the activated T cell and the effect of the activated T cell on the cancer cell is monitored, thereby detecting the anti-cancer cell activity of the target antigenic peptide. It will be appreciated that essentially similar methods are practiced to detect the activity of target antigens from infected cells such as bacterial cells, virally infected cells, parasitically infected cells, or the like. Essentially any protein or peptide is presented to the T cell. For example, in one embodiment, the antigenic peptide is derived from HER-2, and the cancer cell is a breast cancer cell. In another class of embodiments, the antigenic peptide is derived from a protein selected from the group consisting of MART-1 and gp-100, wherein the cancer cell is a melanoma cell. In other embodiments, the antigenic peptide is derived from CEA and the cancer cell is a colon cancer cell. Similar sets of tumor antigens and tumors, or infectious epitopes and infected cells are similarly screened. Depending on the assay format, the activated T cell and target cell are reacted in vitro or in vivo.
Methods of killing a target cell are provided. In the methods, the target cell is contacted with an activated T cell, wherein the T cell is activated by contacting the T-cell with a dendritic cell made from a population of monocytes expanded in the presence of IL-3. The T cells are contacted with the target cell in vitro or in vivo. Typical target cells include infected cells such as bacterially infected, virally-infected, or parasitically infected cells, as well as tumor cells.
Pharmaceutical compositions are provided. The compositions have a pharmaceutically acceptable carrier and a population of at least about 105 dendritic cells, and often at least about 106 cells, occasionally 107 cells, or more, made from a population of monocytes expanded by culture with IL-3, which dendritic cells present a subsequence of a heterologous protein, which population of dendritic cells is competent to activate T cells to kill a target cell in vitro. Typically, the dendritic cells are made from an expanded population of monocytes by incubation with TNF-xcex1, IL-4 and GM-CSF. In one preferred embodiment, the dendritic cells are primarily xe2x80x9cactivatedxe2x80x9d or xe2x80x9ctype IIxe2x80x9d dendritic cells. In some aspects, the population of dendritic cells is competent to activate said T cells against said target cell in vivo. Examples of heterologous protein include HER-2, MART-1, gp-100, PSA, HBVc, HBVs, tyrosinase, MAGE-1, trp-1 and CEA. In other aspects, DC stimulate immune cells such as NK cells, e.g., by secreting cytokines such as IL-12.
Finally, it should be appreciated that the invention provides compositions having a population of isolated monocytes and IL-3. In one preferred embodiment, IL-3 is present at a concentration of about 10 ng/ml.